imgname = ''; %input image name between quotes(e.g. 'img_name.tif') A = imread(imgname); fprintf('------ Image: %s ------\n', imgname); x_size = size(A,2); y_size = size(A,1); A = A * 30; phase = A(:,:,3); data = A(:,:,2); s_test = 0.52:0.005:0.575; results = zeros(1,length(s_test)); figure; for n = -1%[1:length(s_test) -1] if n ~= -1 s = s_test(n); else s = 0.55;%input('Enter best sensitivity: '); end a_thresh = adaptthresh(phase,s); cells = imbinarize(phase, a_thresh); cells = imerode(cells, strel('disk',2,4)); cells = imdilate(cells, strel('disk',6,8)); nw_line = zeros(5,5); nw_line(3,3) = 1; nw_line(4,4) = 1; nw_line( cells = conv2(cells, Remove extremely bright spots cells(data > 3000) = 0; Remove small "cells" cells = bwareaopen(cells,500); cells_red = zeros(y_size,x_size,3); cells_red(:,:,1) = cells; [gx, gy] = gradient(single(phase)); gmax = max(max(gx(:)),max(gy(:))); gmag = sqrt(gx.^2 + gy.^2); gmask = (gmag/gmax) > 0.05; gmask = bwareaopen(gmask, 800); gmask = imclose(gmask,strel('disk',5)); gmask = imerode(gmask,strel('disk',5)); gmask = imdilate(gmask,strel('disk',3)); gmask = imerode(gmask,strel('disk',10)); gmask = imdilate(gmask,strel('disk',10)); gmask = bwareaopen(gmask, 1000); gmask(data > 3000) = 0; gmask_green = zeros(y_size,x_size,3); gmask_green(:,:,2) = gmask; if n ~= -1 subplot(3,4,n); hold on; imagesc(phase); image('CData', cells_red, 'AlphaData', cells*0.3); axis ij; axis tight; title(sprintf('s = %5.3f', s)); colormap gray; end; end; clf; Auto-alignment didn't really work combined = logical(cells); crr_full = xcorr2(single(data), single(combined)); crr = crr_full(((1:y_size) + floor(y_size/2)), ((1:x_size) + floor(x_size/2))); [max_crr, max_crr_i] = max(crr(:)); [max_crr_y, max_crr_x] = ind2sub(size(crr),max_crr_i); x_offset = (max_crr_x) - (x_size/2); y_offset = (max_crr_y) - (y_size/2); x_sclip = (x_offset > 0) * abs(x_offset) + (x_offset <= 0); x_eclip = (x_offset < 0) * abs(x_offset) + (x_offset >= 0); y_sclip = (y_offset > 0) * abs(y_offset) + (y_offset <= 0); y_eclip = (y_offset < 0) * abs(y_offset) + (y_offset >= 0); mask_offset = zeros(y_size,x_size); mask_offset(y_sclip:(y_size-y_eclip),x_sclip:(x_size-x_eclip)) = ... combined(y_eclip:(y_size-y_sclip),x_eclip:(x_size-x_sclip)); mask_offset = combined; cells_green = zeros(y_size,x_size,3); cells_green(:,:,2) = cells_offset; cell_flr = mean(data(mask_offset == 1)); cell_flr_std = std(single(data(mask_offset == 1))); bg_flr = mean(data(mask_offset == 0)); bg_flr_std = std(single(data(mask_offset == 0))); fprintf('Cell Detection Sensitivity : %7.2f\n', s); fprintf('Automatic Mask Offset : (%+3d, %+3d)\n', x_offset, y_offset); fprintf('Cell Fluoresence : %7.2f +/- %7.2f\n', cell_flr, cell_flr_std); fprintf('Background Fluorescence : %7.2f +/- %7.2f\n', bg_flr, bg_flr_std); fprintf('Blank Adjusted Fluoresence : %7.2f +/- %7.2f\n', cell_flr - bg_flr, sqrt(cell_flr_std^2 + bg_flr_std^2)); hold on; subplot(3,2,[1 3]); imagesc(phase); image('CData', cells_red, 'AlphaData', cells*0.2); image('CData', gmask_green, 'AlphaData', gmask*0.2); axis ij; axis tight; title('Cells Image'); colormap gray; subplot(3,2,5); [cells_label, total_cells] = bwlabeln(cells); cell_area = zeros(1,total_cells); for c = 1:total_cells cell_area(c) = bwarea(cells_label == c); end histogram(cell_area,20,'EdgeColor','k','FaceColor','k'); title('"Cell" Size Histogram'); subplot(3,2,[2 4]); hold on; imagesc(data); image('CData', cells_red, 'AlphaData', cells*0.2); image('CData', gmask_green, 'AlphaData', gmask*0.2); axis ij; axis tight; title('Data Image'); set(gca, 'CLim', [800 1400]); colormap gray; subplot(3,2,6); hold on; histogram(data(logical((cells == 0) .* (gmask == 0))),50,'EdgeColor','k','FaceColor','k','Normalization','probability','BinLimits',[0 2000]); histogram(data(cells == 1),50,'EdgeColor','k','FaceColor','r','Normalization','probability','BinLimits',[0 2000]); histogram(data(gmask == 1),50,'EdgeColor','k','FaceColor','g','Normalization','probability','BinLimits',[0 2000]); histogram(data(logical((cells == 1) + (gmask == 1))),50,'EdgeColor','k','FaceColor','y','Normalization','probability','BinLimits',[0 2000]); title('Data Image Histogram'); colormap gray; subplot(3,3,[2 5]); hold on; imagesc(crr); plot([x_size/2 max_crr_x], [y_size/2 max_crr_y], 'r-'); axis ij; axis tight; title('2D Cross Correlation of Data with Cells Position'); colormap gray;